QTL mapping for seed density per silique in Brassica napus

Seed density per silique (SDPS) and valid silique length (VSL) are two important yield-influencing traits in rapeseed. SDPS has a direct or indirect effect on rapeseed yield through its effect on seed per silique. In this study, a quantitative trait locus (QTL) for SDPS was detected on chromosome A09 using the QTL-seq approach and confirmed via linkage analysis in the mapping population obtained from 4263 × 3001 cross. Furthermore, one major QTL for SDPS (qSD.A9-1) was mapped to a 401.8 kb genomic interval between SSR markers Nys9A190 and Nys9A531. In the same genomic region, a QTL (qSL.A9) linked to VSL was also detected. The phenotypic variation of qSD.A9-1 and qSL.A9 was 53.1% and 47.6%, respectively. Results of the additive and dominant effects demonstrated that the expression of genes controlling SDPS and VSL were derived from a different parent in this population. Subsequently, we identified 56 genes that included 45 specific genes with exonic (splicing) variants. Further analysis identified specific genes containing mutations that may be related to seed density as well as silique length. These genes could be used for further studies to understand the details of these traits of rapeseed.


Results
Phenotypic analysis and bulk construction. Phenotypic performance and frequency distribution of SDPS and its related traits in all F 1 plants, F 2 population, F 3 families, and two parental lines were analyzed ( Fig. 1 and Fig.S1). Two parental lines (3001 and 4263) had significantly different phenotypes of SDPS and its related traits; 4263 showed a higher SDPS and more seeds than 3001 in different years, whereas 3001 lines had longer siliques than 4263 lines. The SDPS of F 1 (obtained from 3001 × 4263 cross [2.85 seeds/cm]) and F 1 ′ (obtained from 4263 × 3001 cross [2.72 seeds/cm]) plants was lower than 4263 but higher than 3001, whereas the SPS and VSL of both plants obtained after the reciprocal crosses were significantly higher than those of two parental lines. Both F 2 populations and F 2:3 families demonstrated pronounced phenotypic variations and transgressive segregation of SDPS and its related traits. In the F 2:3 families, the broad-sense heritability estimate of SPDS and VSL were 0.907 and 0.862, respectively, in the parent-offspring regression analysis, indicating that SPDS and VSL were stably inherited. In contrast, SPS demonstrated a broad-sense heritability estimate of 0.293, suggesting sensitivity to environmental variation. Phenotypic data of SDPS, SPS, and VSL showed continuous distributions, and their skewness and kurtosis values were < 1.0 in the segregation populations (Table 1), indicating the quantitative inheritance of these traits. The maximum and minimum SDPS values of F 2 plants were 4.90 seeds/cm and 1.23 seeds/cm, respectively, whereas, at the same time, the SDPS values of 4263 and 3001 parental lines were 4.14 seeds/cm and 2.24 seeds/ cm, respectively (Table 1 and Fig.S1). F 2:3 families also demonstrated a wide distributed SDPS values that ranged from less than that of 3001 to higher than that of 4263. These SDPS data indicated the suitability of subsequent QTL-seq analysis. To construct the high-seed density trait (HT) and low-seed density trait (LT) bulks, F 2 individuals (40 each of HT and LT) were selected. Further, the SDPS of 40 plants in the HT and LT bulks was > 4.01 seeds/cm and < 2.53 seeds/cm, respectively.  (Table 2), based on the Ningyou7 rapeseed genome sequence 12 . In comparison to the reference genome, HT bulk and LT bulk were identified with a total of 5,301,640 and 5,313,578 genome-wide single nucleotide polymorphisms (SNPs) and 1,159,752 and 1,163,307 insertions or deletions (InDels), respectively (Table S1 and Fig. S2).

Identification of SDPS-related genomic region using QTL-seq.
To identify the genomic region associated with SDPS, the Δ(SNP-index) was calculated and plotted against the genome positions based on the information of the SNP index for each bulk (Fig. 2). The Δ(SNP-index) was calculated by subtracting the SNP-   12 . Coincidentally, another QTL, named qSL.A9, was identified for VSL in the same region as qSD.A9-1, explaining a PV of 47.6% in this population. Furthermore, qSD.A9-1 had additive and dominant effects of 0.4 and -0.2, respectively, whereas qSL.A9 had additive and dominant effects of -1.0 and 0.3, respectively, indicating that the major QTL in the region between Nys9A190 and Nys9A531 controlled the expression of SDPS from 4263 and VSL from 3001. We also identified 56 genes in this region predicted based on the annotations of Ningyou7 reference genome 12 (Table S2). Additionally, we detected the following minor QTL: (1) qSD.A9-2 (near qSD.A9-1) for SDPS, with a PV of 10.0% mapped in a 0.96 cM interval between Nys9A503 and Nys9A508, (2) qSP.A9-2 for SPS, which was mapped in the same region as qSD.A9-2 and showed a PV of 7.7%, and (3) qSP.A9-1 for SPS that was located in a 2.16 cM interval between Nys9A136 and Nys9A366 on the same chromosome and accounted for 7.6% of PV ( Fig. 3 and Table 4).

Discussion
Rapeseed silique plays an important role in the accumulation of photosynthetic products, and it was demonstrated that longer siliques with higher seed density could improve the yield of the final products by increasing seed number directly or indirectly 13,14 . Therefore, creating and selecting new varieties with long silique and high seed density may be an effective strategy to increase the rapeseed yield. Previous studies have shown that most agronomic traits in rapeseed, including those related to seeds and siliques, are complex quantitative traits and are controlled by multiple gene products 4,5,14 . Although many QTL for seed weight, SPS, and VSL and underlying genes have been validated and characterized in rapeseed 5,7,14 , only a few QTL or genes are reported for SDPS, the ratio of SL to SPS, in rapeseed. QTL-seq that combines BSA and NGS has been successfully used to rapidly identify many QTL for different traits in rapeseed 6,15,16 . In the present study, QTL that controls SDPS was identified on rapeseed chromosome A09 in a mapping population derived from the 4263 × 3001 crosses. Further, two QTL (qSD.A9-1 and qSL.A9) with 53.1% PV for SDPS from one parent (4263) and 47.6% PV for VSL from the other parent (3001) were mapped on chromosome A09 between 42.22 Mb and 42.63 Mb based on Ningyou7 reference genome sequence 12 . Near this genomic position, one minor QTL for SDPS and two minor QTL for SPS were identified. We speculate the existence of a gene or gene cluster in this region that simultaneously regulates SDPS and VSL or SDPS and SPS. Earlier, a QTL integration analysis performed by Zhou et al. (2021) 14 identified the QTL for SDPS (cqSDPS-A9-2), VSL (cqSL-A9-2), and SPS (cqSPS-A9-2) at the same location on chromosome A09. The physical location of the Table 4. Statistics of seed density within per silique and its related traits QTL identified on chromosome A09 in F 2:3 families. a The distance between the left and right marker of the mapping QTL in F 2:3 families. b PV means the phenotypic variation of each QTL for SDPS or its related trait. c Add is the additive effect. d Dom is the dominant effect.   14 .
To recapitulate the physical location of linkage markers (Nys9A190 and Nys9A531) based on Ningyou7 reference genome 12 , 56 genes were predicted in this region, and 36 of these genes had exonic (splicing) changes. Of all the predicted genes, ChrA09g005075 and ChrA09g005049 were the most likely candidate for qSL.A9 and qSPDS. A9-1, respectively. This is because several previous studies have proved that genes associated with auxin repression affect silique length in Brassica species, and 2OG-Fe(II) oxygenase is a key player in GA and ABA signal transduction 18,19 . GA plays an important role in regulating seed growth and size, while ABA plays an important role in regulating seed number 18 . Among other important genes, those related to functions of seed development are attractive candidates; however, more studies are required to validate their association with SDPS and VSL.

Materials and methods
Plant materials. All seeds of rapeseed accessions used in this study were obtained from the Shanghai Academy of Agricultural Sciences. 4263 had a higher SDPS (more than 3.72 seeds/cm) than that of 3001 (less than 2.24 seeds/cm), while the VSL of 3001 (7.56 cm) was longer than that of 4263 (5.81 cm) in different years. The two rapeseed lines (4263 with short silique but high seed density and 3001 with long silique but low seed density) were selected to develop the mapping population to detect QTL for SDPS and its related traits (Fig. S3). 4263 is a new rapeseed line derived from a self-cross plant of the rapeseed germplasm Su YJ-3, while 3001, which was used as the male, is a new rapeseed line developed from a cross between rapeseed lines Rong Xuan and Jian7. The 4263 × 3001 hybrids were advanced from the F 1 cross by self-cross to yield the segregation population for identifying the QTL of SDPS and its related traits. The phenotypic data of SDPS and its related traits for each F 2:3 families were collected as a mean from the 20 F 3 plants, so as two parental lines. The broad-sense heritability was estimated using parent-offspring regression methods based on the variance of parents, F 1 -derived F 2 , and F 2 -derived F 3 lines 20 .
Sample bulking and DNA isolation. A total of 780 F 2 individuals from 4263 × 3001 crosses were selected to build DNA bulks for QTL-seq. Forty individuals each for HT and LT bulks were selected from the F 2 population based on their extreme phenotypes for SDPS. Genomic DNA from HT bulk, LT bulk, their parents (4263 and 3001), and F 2 individuals were isolated using Plant Genomic DNA Extraction Kit (TIANGEN, China), following the manufacturer's instructions. The quality and concentration of DNA samples were examined by agarose gel electrophoresis (1.5%; w/v).

Illumina sequencing and NGS data analysis.
Test-qualified DNA samples from HT bulk, LT bulk, and two parents (4263 and 3001) were used to construct libraries with an insert size of 350-500 bp using the TruSeq DNA LT Sample Prep Kit and sequenced (150 bp pair-end reads) via an Illumina Xten platform at Shanghai OE Biotech Co., Ltd. (China). Raw data produced from Illumina sequencing were subjected to quality control by Trimmomatic (v0.36) 21 . The filtered clean reads from two DNA bulks and their parents were aligned to the Ningyou7 rapeseed genome 12 using BWA 22 , and SNP calling was performed using SAMtools 23 . The average SNP index was calculated in 1 Mb sliding windows with a 20 kb increment for each pool based on their parent genotype. The △(SNP-index) was calculated by subtracting the SNP-index of LT bulk from that of HT bulk. All SNP-index and △(SNP-index) for all positions were calculated to identify the QTL of SDPS as previously described 6,24 . SSR marker analysis and QTL fine mapping. QTL identified by QTL-seq were validated and finemapped through linkage analysis in F 2:3 families. Primers in the predicted regions were designed using SSR Locator 25 with the parameters as previously described 26 based on the reference genome 12 . All newly developed markers were named as previously described 15 . PCR and amplicon detection in F 2 plants and parental lines were performed with minor modifications as previously described 27 . The polymorphic markers were selected to detect the F 2 population for linkage map construction. Further, the linkage map was drawn using MAP functionality in QTL IciMapping v4.1 28 with the map distance (cM) of Kosambi mapping function 29 . QTL was conducted using the BIP functionality with the ICIM-ADD mapping method in QTL IciMapping v4.1 28 . The LOD threshold and recombination frequency of QTL mapping were set at 3.0 and 0.30, respectively. The designated QTL for SDPS, VSL, and SP was named qSD, qSL, and qSP followed by the chromosome number, respectively. www.nature.com/scientificreports/ Candidate gene analysis of the major QTL for SDPS and VSL. The candidate genes for the major QTL of SDPS and/or VSL were obtained using the Ningyou7 rapeseed genome annotation 12 . Furthermore, the SNPs or InDels in each gene were predicted using SnpEff 30 .

Permission statement.
All the experiments on plant resources, including the collection of rapeseed germplasms, were performed following relevant local guidelines and regulations.

Conclusions
In this study, a total of 780 F 2 lines and 573 F 2:3 families were constructed to elucidate the genetic mechanisms of seed density and its related traits. One major QTL was mapped to a 401.8 kb interval between Nys9A190 and Nys9A531 on rapeseed chromosome A09 and was associated with seed density and silique length in this population based on QTL-seq and linkage analysis. The PV of SDPS and VSL was 53.1% and 47.6%, respectively, in F 2:3 families. These findings provide a basis to conduct genetic breeding experiments involving cloning of both seed density and silique length in rapeseed.